| Soybean [Glycine max(Linn.)Merrill],which is among the most important legume crops,and the world's highest-yield oil crop.Heat and drought are two major abiotic stressors limiting soybean qualities and output.In order to define the mechanism of drought(D),high temperature(H)and drought and high temperature combined stress(DH)on soybean.88 varieties of cultivated soybeans were evaluated for drought tolerance at the bud stage and seedling stage,and the drought-tolerant variety Heinong 44 was finally selected as the follow-up research material.The potted method was used to process 0,1,3,6,8,12,24,28,32 and 48 h of D,H and DH treatments at soybean seedling stsge(V3).Based on the variation trend in the physiological indexes during all treatments and plant phenotypic under stress,the 8 h and 24 h treatments were selected for the subsequent studies of transcriptional expression profiling and proteome,0h was as the control.RNA-seq sequencing analysis was performed on BGISEQ-500 platform,the response mechanism of soybean to D,H and DH stresses has been discussed at transcriptional level.The effects of drought stress on soybean leaf tissue proteome were analyzed by using proteomics IBT technique,and further analysis of how soybeans respond to drought stress at the transcriptional level and protein level were done.The main results are as follows:1.Through comprehensive evaluation of drought tolerance of soybean at bud stage and seedling stage,a total of 6 drought tolerant varieties,12 relatively drought tolerant varieties,24 intermediate type varieties,38 relatively drought sensitive varieties,and 8 drought sensitive varieties were screened out at bud stage.And a total of 9 drought tolerant varieties,23 relatively drought tolerant varieties,28 intermediate type varieties,14 relatively drought sensitive varieties,and 14 drought sensitive varieties were screened out at seedling stage.2.The detection of physiological indicators under drought,high temperature and combined stress found that the SOD activity,the content of soluble sugar,MDA and proline all raised,and the increase of DH was higher than D after 8h,but less than H.3.21 digital expression profiles under drought,high temperature and combined stress treatments were constructed through high-throughput sequencing of leaf tissues.18 and 93up-regulated and 36 and 85 down-regulated key drought response genes associated with stress response,defense response,and transcriptional regulation were identified at drought stress 8 hand 24 h.Meanwhile,120 and 132 up-regulated and 53 and 23 down-regulated key high temperature response genes were identified at high temperature stress 8 h and 24 h,and these genes were mainly involved in functions such as stress response,protein binding,and transmembrane transportation.4.The analysis showd that although most of(55.82-61.90%)the differential expression genes had the same to D or H,but still 1021(44.18% of upregulated genes)and 544(38.10% of downregulated genes)genes exhibited specific response to DH.These responsive gens and metabolic pathways were more similar to H and were different from D.Further GO enrichment analysis confirmed our observation that,metabolic process was commonly enriched by downregulated genes of three treatments at both 8 h and 24 h.Generation of precursor metabolites and energy were commonly enriched at 8 h,and oxidation reduction was commonly enriched at 24 h by downregulated genes of three treatments(D,H and DH).Furthermore,more functions and genes exhibited specificity in the combination of heat and drought stress.For example,cell redox homeostasis and cellular homeostasis were enriched specifically at 8 h and cellular respiration and extracellular polysaccharide metabolic process were enriched at 24 h by upregulated genes of combination stress.Defense response was enriched at 8 h and steroid biosynthetic process was enriched at 24 h specifically by downregulated genes of combination stress.5.We identified 147 stress response transcription factors distributed among 34 transcription factor families under D,H and DH stress treatments,and could be classified into 9 cluster based on their expression patterns with K-mean cluster analysis.Among them,11 TF families accounted for three-quarters of stress-responsive TF genes,including AP2-EREBP ? bHLH ?MYB?HSF?C2C2-Dof?G2-like?NAC?WRKY?bZIP?TCP?zf-HD.And AP2-EREBP,bHLH and MYB were the most widely involved in the response of soybean to drought and high temperature stresses.6.We identified four proline-related differentially expressed genes under drought stress,which were upregulated at 8 h and 24 h.Three differentially expressed genes related to SOD were identified,one of them was annotated to the SOD Cu-Zn family,which was downregulated at 8h,and upregulated at 24 h,and another two were annotated to the SOD Fe-Mn family,which were downregulated at 8 h and 24 h.In addition,differentially expressed genes associated with peroxidase,glutathione peroxidase and catalase were identified as 12,4 and 1 respectively.The expression patterns of these genes were consistent with the trend of SOD activity and proline content under drought stress.7.65 differentially expressed genes of abscisic acid,gibberellin,auxin,cytokinin,jasmonic acid,salicylic acid,brassinosteroid and ethylene signal transduction pathway were obtained under drought stress,including important drought-resistant genes such as DELLA,SAUR,PP2 C,MYC2,etc.In addition,CNC co-expression analysis of differentially expressed genes and transcription factors of plant hormone signal transduction under drought stress revealed thattranscription factor MYB173 was the core transcription factor,and it played an important role in the regulation of plant hormone signaling pathways.8.5183 proteins were found by the protein sequencing of leaf tissue under drought stress,where 75 and 320 up-regulated differential proteins,70 and 84 down-regulated differential proteins were obtained at 8 h and 24 h,respectively.Further GO enrichment analysis showed that the differentially expressed proteins did not only involve in catalytic activities,binding and other molecular functions,but also involved in metabolic processes,cellular processes and other biological processes.And it found that there were 12 photosynthesis-related differentially expressed proteins,such as chlorophyll a-b binding protein,ribulose bisphosphate carboxylase,etc.;14 differentially expressed proteins related to carbon metabolism,such as enolase and fructose diphosphate aldolase,etc.;22 differentially expressed proteins related to energy metabolism,such as ferredoxin and carbonic anhydrase,etc.9.The classification and functional analysis of identified differentially expressed proteins showed that 549 differential proteins were identified in drought stress treatment at 8h and 24 h,and they had 13 significantly enriched metabolic pathways.Among them,8 metabolic pathways were enriched at 8h in drought stress,including photosynthesis-antenna protein and linoleic acid metabolism were upregulated expression,phenylpropanoid biosynthesis,stilbene diarylheptanoid and gingerol biosynthesis,pyrimidine metabolism,flavonoid biosynthesis,inositol phosphate metabolism and phenylalanine metabolism were downregulation expression.5 metabolic pathways were enriched at 24 h in drought stress,including ribosome,ribosome biogenesis in eukaryotes and Valine,leucine and isoleucine degradation were upregulated expression,and downregulation expression of glutathione metabolism and inositol phosphate metabolism.10.The transcriptome and proteome correlation analysis showed that linoleic acid9s-lipoxygenase and mannan endo-1,4-beta mannosidase was co-responsive gene/protein at 8 h and 24 h under drought stress.The associated differentially expressed genes/proteins weremainly involved in some functions,such as protein binding and nucleic acid binding functions;oxidoreductase activity and hydrolase activity functions;protein metabolism processes and lipid metabolism processes;abiotic stimulus response and hormone respons,etc. |
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